Hydrophobic taxane derivatives

ABSTRACT

This invention provides a taxane derivative of the formula:wherein a hydrophobic organic moiety is attached to a taxane. R and R1 is each indepently H or a hydrophobic organic moiety, as long as at least one of R and R1 is not H. Attachment of a hydrophobic organic moiety to the taxane so as to obtain a taxane derivative generally stabilizes the association of the derivative with a lipid, including a liposomal lipid, carrier in the plasma of animals to which the derivative-carrier association is administered. Also provided herein is a composition containing the taxane derivative and a pharmaceutically acceptable medium; desirably, the medium also contains a lipid carrier, and the derivative is associated with the carrier. Further provided herein is a method of administering taxane derivatives to animals, for example, animals afflicted with cancers.

This application is a continuation of U.S. Ser. No. 08/753,650, filedNov. 27, 1996, now U.S. Pat. No. 6,118,011 which is a continuation ofU.S. Ser. No. 08/474,888, filed Jun. 7, 1995, now U.S. Pat. No.5,580,899, which is a continuation-in-part of U.S. Ser. No. 08/369,817,filed Jan. 9, 1995, now abandoned.

This invention provides a taxane derivative comprising a hydrophobicorganic moiety attached to a taxane, compositions comprising suchcompounds, including lipid carrier-containing compositions, and methodsof administering such compositions to animals, including those afflictedwith cancers.

Taxanes can be anticancer agents, which affect cell growth by blockingcell division. Paclitaxel, for example, is an antimitotic agent whichbinds to tubulin, thereby blocking the disassembly of microtubules andconsequently, inhibiting cell division (Schiff et al., Nature 277:665(1979)). The optimal effect of paclitaxel on polymerization andstabilization of microtubuies is seen at concentrations nearstoichiometric equivalence with tubulin dimers (Schiff and Horowitz,Proc. Natl. Acad. Sci. USA 77(3):1561-1565(1980)). Paclitaxel has beenfound to have activity against ovarian and breast cancers, as well asagainst malignant melanoma,. colon cancer, leukemias and lung cancer(see, e.g., Borman, Chemical & Engineering News, Sep. 2, 1991, pp.11-18; The Pharmacological Basis of Therapeutics * * * (Goodman Gilmanet al, eds.) * * * , Pergamon Press, New York (1990), p. 1239; Suffness,Antitumor Alkaloids, in: “The Alkaloids, Vol. XXV,” Academic Press, Inc.(1985), Chapter 1, pp. 6-18; Rizzo et al., J. Pharm. & Biomed. Anal.8(2): 159-164 (1990); and Biotechnology 9:933-938 (October 1991).

Paclitaxel can be isolated from natural sources, or preparedsynthetically frpm naturally occurring precursors, e.g., baccatin, byattachment of protecting groups to the hydroxyl groups of theseprecursors that are to become the hydroxyl groups of paclitaxel,converting the precursors, and then removing the protecting groups fromthe hydroxyl groups to obtain paclitaxel (see, e.g., WO93/10076, int.pub. date May 27, 1993; K. V. Rao, U.S. Pat. No. 5,200,534; R. A.Holton, U.S. Pat. No. 5,015, 744; PCT/US92/07990; V. J. Stella and A. E.Mathew, U.S. Pat. No. 4,960,790; K. C. Nicolau, Nature 364 (1993), pp.464-466; Nicolaou, K. C. et al. Nature 367 (1994) pp.630-634; Holton, R.A., et al. J. Am. Chem. Soc. 116 (1994) pp. 1597-1600; WO93/16059, int.pub. date Aug. 19, 1993; EP 528,729, published Feb. 24, 1993; EP522,958, published Jan. 13, 1993; WO91/13053, int. pub. date Sep. 5,1991; EP 414,610, int. pub. date Feb. 27, 1991). The protecting groupsused in some of these synthetic processes are short-chain aliphaticalkyl groups, but are not hydrophobic organic moieties as the term isused herein.

Paclitaxel is highly insoluble in water and aqueous solvents, and iscurrently supplied as an emulsion (TAXOL®, Bristol-Myers Squibb) in apolyoxyethylated derivative of castor oil and ethanol (CremophorEL®).However, administration of this formulation generally entailspremedication with other drugs and a slow infusion of a large volume, toavoid toxicity associated with the Cremophor vehicle. Patients aretherefore generally required to be admitted to hospitals over night.Compositions provided herein comprising a taxane derivative associatedwith a lipid carrier can solve this problem, by providing a formulationin which the taxane remains stably associated with the lipid carrierwhen administered. Stable association with a lipid carrier generallyavoids the toxicity problems encountered with the currently useddelivery system, as well as the need for slow-infusion administration.

SUMMARY OF THE INVENTION

This invention provides a taxane derivative of the formula:

wherein: A¹ is H or a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)—. Q is C₆H₅—, (CH₃)₃CO— or (CH₃)CH═C(CH₃)—;A² is H or CH₃C(O)—; A³ is H, or OH. A¹ is preferably(C₆H₅)(O)NHCH(C₆H₅)CH(OR)C(O)—. Preferably, A² is CH₃C(O)—, and A³ is Hthat is, the taxane derivative preferably is a paclitaxel.

When R¹ is H, A¹ is a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)—; R is then not H, but rather, is a grouphaving the formula Y¹, Z¹X¹ or Z¹D¹. When A¹ is H, or when A¹ is a grouphaving the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)— and R is H, R¹ is thennot H; rather, R¹ is then a group having the formula Y², Z²X² or Z²D².Accordingly, at least one hydrophobic organic moiety is attached to thetaxane. Furthermore, two hydrophobic organic moieties can be attached tothe taxane, R then being a group having the formula Y¹, Z¹X¹ or Z¹ D¹when R¹ is a group having the formula Y², Z²X² or Z²D².

Each of Y¹ and Y² is independently a group having the formula:—C(O)(CH₂)_(a)(CH═CH)_(b)(CH₂)_(c)(CH═CH)_(d)(CH₂)_(e)(CH═CH)_(f)(CH₂)_(g)(CH═CH)_(h)(CH₂)_(i)CH₃.The sum of a+2b+c+2d+e+2f+g+2h+i is equal to an integer from 7 to 22(refering to the number of carbon atoms); a is zero or an integer from 1to 22; each of b, d, f and h is independently zero or 1; c is zero or aninteger from 1 to 20; e is zero or an integer from 1 to 17; g is zero oran integer from 1 to 14; i is zero or an integer from 1 to 11; and a toi can be the same or different at each occurrence.

Each of Z¹ and Z² is independently a linker of the formula:—C(O)(CH₂)_(j)(CH═CH)_(k)(CH₂)_(l)(CH═CH)_(m)(CH₂)_(n)(CH═CH)_(o)(CH₂)_(p)(CH═CH)_(q)(CH₂)_(r)C(O)—.The sum of j+2k+l+2m+n+2o+p+2q+r is equal to an integer from 2 to 22;each of k, m, o and q is independently zero or 1; j is zero or aninteger from 2 to 22; l is zero or an integer from 1 to 20; n is zero oran integer from 1 to 17; p is zero or an integer from 1 to 14; and r iszero or an integer from 1 to 11. Each of j to r can be the same ordifferent at each occurrence.

Each of X¹ and X² is independently a group having the formula:

G¹ is —OP(O)₂OCH₂CH₂N(CH₃)₃ (phosphorylcholine), —OP(O)₂OCH₂CH₂NH₂,(phosphorylethanolamine) —OP(O)₂OCH₂CH(OH)CH₂OH (phosphorylglycerol),—OP(O)₂OCH₂CH(NH₂)CO₂H (phosphorylserine) or phosphoylinositol.

Each of D¹ and D² is independently a group having the formula:

When R is not H, it is preferably a group having the formula Y¹. Y¹ispreferably a group having the formula —C(O)(CH₂)_(a)CH₃, and still morepreferably, is —C(O)(CH₂)₁₀CH₃ or —C(O)(CH₂)₁₆CH₃. However, R can alsobe a group having the formula Z¹X¹. G¹ is then preferablyphosphorylcholine, Z¹ is preferably —C(O)(CH₂)₈C(O)— and R is preferablya group having the formula:

wherein Y¹ is preferably a group having the formula —C(O)(CH₂)_(a)CH₃.

R can further be Z¹ D¹. Z¹ is then preferably a group having the formula—C(O)(CH₂)_(j)C(O)—, more preferably, —C(O)(CH₂)₃C(O)—.

When R¹ is not H, it is preferably Y2. More preferably, R¹ is then agroup having the formula —C(O)(CH₂)_(a)CH₃, and still more preferably,—C(O)(CH₂)₁₀CH₃ or —C(O)(CH₂)₁₆CH₃. However, R¹ can then also be a grouphaving the formula Z²X²; G¹ is then preferably phosphorylcholine and Z²is preferably —C(O)(CH₂)₈C(O)—. R¹ can further be a group having theformula Z²D²; Z² is then preferably a group having the formula—C(O)(CH₂)_(j)C(O)—, more preferbly, —C(O)(CH₂)₃C(O)—.

Hydrophobic organic moieties can be attached to the same taxane at boththe 2′ and 7 positions; neither R nor R¹ is then H. R and R¹ can both bethe same moiety, such as groups having the formula —C(O)(CH₂)₁₀CH₃ or—C(O)(CH₂)₁₆CH₃, or different moieties, but are preferably the samemoiety.

Also provided herein is a composition comprising a pharmaceuticallyacceptable medium and the taxane derivative of this invention. Themedium preferably comprises a lipid carrier, for example, a fatty acid,lipid, micelle, aggregate, lipoprotein or liposome, associated with thetaxane. Preferably, the lipid carrier is a liposome. The lipid carriercan comprise an additional bioactive agent, that is, a bioactive agentin addition to the taxane derivative. Lipid carriers can also comprise aheadgroup-modified lipid.

Further provided herein is a method of administering a taxane derivativeto an animal, preferably a human. The method of this invention can beused to treat an animal afflicted with a cancer, by administering to theanimal an anticancer effective amount of the derivative. “Anticancereffective” amounts of a taxane derivative are typically at least about0.1 mg of the derivative per kg of body weight of the animal to whichthe derivative is administered; generally, the anticancer effectiveamount of the taxane is from about 0.1 mg per kg of body weight to about1000 mg/kg. Preferably, the anticancer taxane derivative administered isa paclitaxel derivative.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Histograms reflecting association of paclitaxel and paclitaxelhydrophobic organic moiety conjugates with palmitoyloleoyl (POPC)liposomes. X-axis: paclitaxel; 2′—C12 paclitaxel (—C(O)(CH₂)₁₂CH₃attached to paclitaxel at the 2′ position); 7—C12 paclitaxel(—C(O)(CH₂)₁₂CH₃ attached to paclitaxel at the 7 position) and 2x—C12paclitaxel (—C(O)(CH₂)₁₂CH₃ attached to paclitaxel at the 2′ and 7positions). Y-axis: percentage of paclitaxel associated with liposomes.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a taxane derivative of the formula:

comprising a hydrophobic organic moiety attached to a taxane. A¹ is H ora group having the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)—. Q is C₆H₅—,(CH₃)₃CO—, or CH₃CH═C(CH₃)—; A² is H or CH₃C(O)—; A³ is H or OH.

A¹ is preferably a group having the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)—;Q is then preferably C₆H₅, A² is then preferably CH₃C(O)— and A³ is thenpreferably H. Accordingly, paclitaxel ([Compound I]); TAXOL® (C₄₇H₅₁NO),Bristol-Myers Squibb) is preferred herein. However, taxotere (II), whichdiffers from paclitaxel by having a tert-butoxy carbonyl group at theC-13 position, instead of a benzoyl group, and a hydroxyl group, insteadof an acetyloxy group, at C-10 is also useful herein. Accordingly, fortaxotere, A¹ is (CH₃)₃COC(O)NHCH(C₆H₅)CH(OR)(O)—, A² is H, and A³ is H.Cephalomannine (III), differs from paclitaxel in the amide group locatedat the distal end of the C-13 ester. A¹ is then(CH₃)CH═C(CH₃)C(O)NHCH(C₆H₅)CH(OR)C(O)—, A² is CH₃C(O)—and A³ is H.Additional taxanes useful in accordance with the practice of thisinvention are known in the art and include, without limitation:19-hydroxybaccatin III [IV] (McLaughlin, et al., J. Nat. Prod.,44:312-319 (1981)), Baccatin V [V], 10-deacetyl cephalomannine [VI],10-deacetyl paclitaxel [VII], 7-Epi-10-deacetyl paclitaxel [VIII],7-Epi-10deacetyl cephalomannine [IX], and 10-deacetyl baccatin III [X],as described in the following table, in addition to paclitaxel, taxotereand cephalomannine. The compound names listed are for unsubstituted, or“free”, taxanes, that is, taxanes to which hydrophobic organic moietiesare not attached.

Compound A¹ A² A³ Paclitaxel (I) C₆H₅C(O)NHCH(C₆H₅) CH₃C(O)— HCH(OR)C(O)— Taxotere (II) C(CH₃)₃OC(O)NHCH H H (C₆H₅)CH(OR)C(O)—Cephalo- (CH₃)CH═C(CH₃)C(O)NHCH CH₃C(O)— H mannine (III)(C₆H₅)CH(OR)C(O)— 19-hydroxy H CH₃C(O)— OH baccatin III (IV) BaccatinIII (V) H CH₃C(O)— H 10-Deacetyl (CH₃)CH═C(CH₃)C(O) H H cephaloNHCH(C₆H₅)CH(OR)C(O)— mannine (VI) 10-Deacetyl C₆H₅C(O)NHCH(C₆H₅) H Htaxol CH(OR)C(O)— (VII) (7α-OH) 7-Epi-10- C₆H₅C(O)NHCH(C₆H₅) H Hdeacetyl CH(OR)C(O)— taxol(7β-OH) (VIII) 7-Epi-10- (CH₃)CH═C(CH₃)C(O) HH deacetyl NHCH(C₆H₅)CH(OR)C(O)— cephalo mannine(7β- OH) (IX)10-Deacetyl H H H baccatin III (X)

R and R¹ can each independently be either H or a hydrophobic organicmoiety, as long as at least one of R and R¹ is not H. “Hydrophobicorganic moieties” are carbon-based molecular groups which can beattached to taxanes. Free taxanes can readily dissociate from lipidswith which they have been associated in the plasma of animals to whichthe taxane/lipid associations have been administered. Attachment of ahydrophobic organic moiety to a free taxane so as to obtain a taxanederivative can stabilize the association of the derivative with a lipid.

Hydrophobic organic moieties include, without limitation, saturated orunsaturated, aliphatic or branched fatty acids. Such moieties alsoinclude: polyol-, e.g., glycerol or mannitol, based amphipathic lipidscomprising a polar group and one or more fatty acids. Furthermore, otherhydrophobic organic moieties, including sphingolipids such assphingomyelin, which can stabilize the association between a taxanederivative and a lipid in an animal's plasma can also be attached to ataxane according to the practice of this invention; selection of suchother moieties is within the purview of ordinarily skilled artisansgiven the teachings of this invention.

“Attachment” means conjugation, covalent binding, linking, conjugationor otherwise forming a chemical connection between a taxane and ahydrophobic organic moiety. Attachment of the moiety is to one or morereactive groups, typically hydroxyl groups, on the taxane. Paclitaxel,for example, has three hydroxyl groups to which hydrophobic organicmoieties can be attached. These are located at the 2′, 7 and 1positions, with their relative order of reactivity generally believed tobe (from most reactive to least reactive) 2′>7>>1. Hydrophobic organicmoieties can be attached to the primary reactive group of a taxane,e.g., the 2′ OH group of paclitaxel, utilizing stoichiometric amounts ofthe moiety to be attached, e.g., fatty acid chlorides or anhydrides.Reactions are typically performed in the presence of a base, such aspyridine, dimethylaminopyridine, triethylamine, or others, and incommonly used polar, aprotic organic solvents. The progress of thereaction, at room temperature, can be monitored by a number of wellknown chromatographic means, for example, thin layer chromatographyusing a 3% methanol-in-chloroform solvent system. The compound'sidentity can be confirmed by spectroscopic and other analyticalprocedures, such as NMR spectroscopy.

Specific reaction and purification conditions are generally expected tovary according to a number of factors, including without limitation, theraw materials and reactants used, that are well within the purview ofordinarily skilled artisans to determine and control given the teachingsof this invention. For example, for the attachment of lauric acid (C12)to paclitaxel, 9 mg (0.074 mmoles) dimethylaminopyridine (DMAP), 50 mg(0.059 mmole) of paclitaxel and 15 mg (0.068 mmoles) lauroyl chloridecan be combined with 5 ml of chloroform.

Attaching hydrophobic organic moieties to less reactive groups on thetaxane typically requires use of an amount of an active form of themoiety that is in excess of the stoichiometric amount. The hydroxylgroup at the 7 position of paclitaxel, for example, can be modified, forexample, by attaching a hydrophobic organic moiety to both the 2′ and 7OH groups, and then selectively removing the 2′ moiety, such that themoiety at the 7 position remains attached to paclitaxel. Such reactionscan be performed using essentially the same procedures as thosedescribed above. Selective removal of the 2′ modification can beaccomplished using stoichiometric amounts of a mild base, e.g., sodiumbicarbonate.

Additionally, the 7 OH group of paclitaxel can be modified by“protecting” the 2′ OH group before covalently linking the drug with thehydrophobic organic moiety. The 2′ OH group can also be protected withgroups such as, for example, triphenyl methyl, methoxytriphenyl methyl,trifluoroacetyl and hexanoyl groups, using processes generally known toordinarily skilled artisans. The protected paclitaxel is then reactedwith an active form of the moiety, e.g., fatty acid anhydrides orchlorides, in anhydrous organic solvent and bases such as DMAP andpyridine. The protecting group can be removed from the 2′ position bywell known and readily practiced means, under mildly acidic or basicconditions. Lauric acid can, for example, be attached to the 7 OH groupof paclitaxel by combining 54 mg (0.44 mmoles) DMAP, 50 mg (0.059mmoles) paclitaxel, and 77 mg (0.35 mmoles) of lauroyl chloride with 5ml of chloroform, keeping the reaction at room temperature, so as toobtain 2′7-dilauroyl paclitaxel. Then, 3.0 mg NaCl in 75 microliters ofwater can be added to a solution of chloroform/methanol (1:1) containing58 mg (0.048 mmoles) of 2′7-dilauroyl paclitaxel to remove the lauricacid attached to the 2′-OH group. This reaction can be incubated at 30degrees C. and followed closely by thin layer chromatography (TLC).Attachment, however, is not limited to use of these specific amounts;rather, ordinarily skilled artisans can vary the amounts for reasons,and within ranges, well known to them, given the teachings of thisinvention.

R can be H or a group having the formula Y¹, Z¹X¹ or Z¹D¹; R¹ can be H,or a group having the formula Y², Z²X² or Z²D². At least one of R and R¹is not H. Y¹, Z¹X¹, Z¹D¹, Y², Z²X² and Z²D² are hydrophobic organicmoieties.

Each of Y¹and Y² is independently a group having the formula:—C(O)(CH₂)_(a)(CH═CH)_(b)(CH₂)_(c)(CH═CH)_(d)(CH₂)_(e)(CH═CH)_(f)(CH₂)_(g)(CH═CH)_(h)(CH₂)_(i)CH₃.The sum of a+2b+c+2d+e+2f+g+2h+i is equal to an integer from 7 to 22(refering to the number of carbon atoms); a is zero or an integer from 1to 22; each of b, d, f and h is independently zero or 1; c is zero or aninteger from 1 to 20; e is zero or an integer from 1 to 17; g is zero oran integer from 1 to 14; i is zero or an integer from 1 to 11; and a toi can be the same or different at each occurrence. Preferably, each ofY¹and Y² is independently saturated, that is, there are no double bondsbetween adjacent carbon atoms. Accordingly, b, d, f and h are eachpreferably zero, c, e, g, and i are each also zero, and Y¹and Y² areeach independently groups having the formula —C(O)(CH₂)_(a)CH₃, whereina is an integer from 7 to 22. More preferably, each of Y¹ and Y² isindependently —C(O)(CH₂)₁₀CH₃ or —C(O)(CH₂)₁₆CH₃. Alternatively, Y¹ andY² can each be unsaturated, that is, they can have one or more CH═CHgroups. In this case, at least one of b, d, f or h is not zero. Forexample, when the unsaturated hydrocarbon has one double bond: b is 1,d, f and h being zero; Y¹and Y² are each then independently a grouphaving the formula —C(O)(CH₂)_(a)CH═CH(CH₂)_(C)CH₃; a is zero or aninteger from 1 to 18; c is also zero or an integer from 1 to 18, atleast one of a or c is not zero, and the sum of a and c is equal to aninteger of from 5 to 20.

X¹ and X² are each independently a group having the formula:

G¹ is preferably a phosphate-based polar group, including withoutlimitation: —OP(O)₂OCH₂CH₂N(CH₃)₃ (phosphorylcholine), —OP(O)₂OCH₂CH₂NH₂(phosphorylethanolamine) —OP(O)₂OCH₂CH(OH)CH₂OH (phosphorylglycerol),—OP(O)₂OCH₂CH(NH₂) CO₂H (phosphorylserine) and phosphorylinositol. Morepreferably, G¹ is phosphorylcholine. However, nitrogen, sulfur and otheratoms can be substituted for the phosphorous. Y¹ is preferably a grouphaving the formula —C(O)(CH₂)_(a)CH₃.

Each of Z¹ and Z² is independently a linker of the formula:—C(O)(CH₂)_(j)(CH═CH)_(k)(CH₂)_(l)(CH═CH)_(m)(CH₂)_(n)(CH═CH)_(o)CH₂)_(p)(CH═CH)_(q)(CH₂)_(r)C(O)—.The sum of j+2k+l+2m+n+2o+p+2q+r is equal to an integer of from 2 to 22;each of k, m, o and q is independently zero or 1; j is zero or aninteger from 2 to 22; l is zero or an integer from 1 to 20; n is zero oran integer from 1 to 17; p is zero or an integer from 1 to 14; and r iszero or an integer from 1 to 11. Each of j to r can be the same ordifferent at each occurrence. Preferably, each of Z¹ and Z² isindependently a group having the formula —C(O)(CH₂)_(j)C(O)—, morepreferably, each of Z¹ and Z² is —C(O)(CH₂)₈C(O)—.

D¹ and D² are each independently:

Y¹ and Y² are then preferably and independently each a group having theformula —C(O)(CH₂)_(a)CH₃. For example, both Y¹ and Y² can each be—C(O)(CH₂)₁₄CH₃.

When R¹ is H, A¹ is a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)—, and R is not H. R is then a group havingthe formula Y¹, Z¹X¹, or Z¹D¹, and the taxane derivative comprises ahydrophobic organic moiety attached at the 2′ position of the taxane.When A¹ is H, or when A¹ is a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O— and R is H, then R¹ is not H. R¹ is then Y²,Z²X² or Z²D², and the taxane derivative has a hydrophobic organic moietyattached at the 7 position. Alternatively, the taxane derivative canhave a hydrophobic organic moiety attached at both the 2′ and the 7positions of the taxane; these moieties can be the same or different ateach occurence, but are preferably the same. R is then a group havingthe formula Y¹, Z¹X¹, or Z¹D¹ when R¹ is a group having the formula Y²,Z²X², or Z²D².

Also provided herein is a composition comprising the taxane derivativeof this invention and a pharmaceutically acceptable medium; such amedium preferably comprises a lipid carrier associated with the taxanederivative. “Pharmaceutically acceptable media” are generally intendedfor use in connection with the administration of active ingredients toanimals, for example, humans, and include solids, such as pills,capsules and tablets, gels, excipients or aqueous or nonaqueoussolutions. Active ingredients can, for example, be combined with,dissolved, suspended, or emulsified in or with such media.Pharmaceutically acceptable media are generally formulated according toa number of factors well within the purview of the ordinarily skilledartisan to determine and account for, including without limitation: theparticular active ingredient used, its concentration, stability andintended bioavailability; the disease, disorder or condition beingtreated with the composition; the subject, its age, size and generalcondition; and the composition's intended route of administration, e.g.,nasal, oral, ophthalmic, topical, transdermal, vaginal, subcutaneous,intramammary, intraperitoneal, intravenous, or intramuscular (see, forexample, J. G. Nairn, in: Reminaton's Pharmaceutical Science (A.Gennaro, ed.), Mack Publishing Co., Easton, Pa., (1985), pp. 1492-1517).Typical pharmaceutically acceptable media used in parenteral drugadministration include, for example, D5W, an aqueous solution containing5% weight by volume of dextrose, and physiological saline.Pharmaceutically acceptable media can contain additional ingredientswhich enhance the stability of the active ingredients, includingpreservatives and anti-oxidants.

A “lipid carrier” is a a hydrophobic substance, or an amphipoathicsubstance having a hydrophobic domain, with which the taxane derivativeof this invention can form a stable association, and which is suitablefor therapeutic administration to animals. “Association” as used hereingenerally means association between the hydrophobic organic moietyattached to the taxane and the hydrophobic portion of the lipid carrier.Hydrophobic organic moieties and hydrophobic lipid domains generallyassociate through the action of a number of forces, such as Van derWaal's forces, generally known to operate between hydrophobic moleculesin an aqueous environment. Means of determining the stability of suchassociations, for example, by determining the percentage of taxanederivative recoverable with phosphorous when the lipid carrier comprisesa phospholipid, are well known to, and readily practiced by, ordinarilyskilled artisans given the teachings of this invention. Ordinarilyskilled artisans can, given the teachings of this invention, selectsuitable lipid carriers. These include, without limitation: fatty acids,amphipathic lipids, liposomal or nonliposomal, lipoproteins and others.Preferably, the lipid carrier with which the taxane derivative of thisinvention is associated is a liposome.

Liposomes comprise one or more bilayers of lipid molecules, each bilayerencompassing an aqueous compartment. Unilamellar liposomes have a singlelipid bilayer and multilamellar liposomes have more than one bilayer(for a review see, for example, see Chapman, “Physicochemical Propertiesof Phospholipids and Lipid-Water Systems,” in: Liposome Technology,Volume I: Preparation of Liposomes (G. Gregoriadis, ed.). CRC Press,Boca Raton, Fla. (1984), pp. 1-18, the contents of which areincorporated herein by reference). The amphipathic lipid molecules whichmake up lipid bilayers comprise a polar (hydrophilic) headgroup and oneor two acyl chains. The polar groups can be phosphate-, sulfate ornitrogen-based groups, but are preferably phosphate groups, such asphosphorylcholine, phosphorylethanolamine, phosphorylserine,phosphorylglycerol or phosphorylinositiol. The fatty acids generallycomprise from 4 to 24 carbon atoms, and can be saturated (e.g.,myristic, lauric, palmitic, or stearic acids, or unsaturated (e.g.,oleic, linoleic, linoleic and arachidonic acid). Furthermore, liposomescan also comprise sterols, such as cholesterol, and other lipids.

Liposomes can be made by a variety of methods, including: Bangham'smethods for making muiltilamellar liposomes (MLVs); Lenk's, Fountain'sand Cullis' methods for making MLVs with substantially equalinterlamellar solute distribution; extrusion, sonication orhomogenization of MLVs to make unilamellar liposomes; and ether orethanol injection processes (see, for example, U.S. Pat. Nos. 4,522,803,4,588,578, 5,030,453, 5,169,637 and 4,975,282, and R. Deamer and P.Uster, “Liposome Preparation: Methods and Mechanisms,” in Liposomes (M.Ostro, ed.), Marcel Dekker, Inc., New York (1983), pp. 27-52, thecontents of which are incorporated herein by reference).

Lipid carriers associated with the taxane derivative of this invention,for example, liposomes, can comprise an additional bioactive agent, thatis, a bioactive agent in addition to the taxane derivative. Liposomes,for example, can be loaded with biologically active agents bysolubilizing the agent in the lipid or aqueous phase used to prepare theliposomes. Alternatively, ionizable bioactive agents can be loaded intoliposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionizable agent to theaqueous medium external to the liposome (see Bally et al. U.S. Pat. No.5,077,056, and U.S. Ser. No. 08/112,875, the contents of which areincorporated herein by reference).

Lipid carrier/bioactive agent formulations can enhance the therapeuticindex of the bioactive agent, for example by buffering the agent'stoxicity and by reducing the rate at which the agent is cleared from thecirculation of animals, thereby meaning that less of the agent need beadministered to achieve the desired therapeutic effect. In this regard,lipid carriers, for example, liposomes, can also comprise one or moreheadgroup-modified lipids, which are amphipathic lipids whose polarheadgroups have been derivatized by attachment thereto of a chemicalmoiety, e.g., polyethylene glycol, a polyalkyl ether, a ganglioside, anorganic dicarboxylic acid or the like, which can inhibit the binding ofserum proteins to lipid carriers such that the pharmacokinetic behaviorof the carriers in the circulatory systems of animals is altered (see,e.g., Blume et al., Biochim. Biophys. Acta. 1149:180 (1993); Gabizon etal., Pharm. Res. 10(5):703 (1993); Park et al. Biochim. Biophys Acta.1108:257 (1992); Woodle et al., U.S. Pat. No. 5,013,556; Allen et al.,U.S. Pat. Nos. 4,837,028 and 4,920,016; U.S. Ser. No. 142,691, filedOct. 25, 1993). Lipid carriers are generally cleared from animals'circulations by their reticuloendothelial systems (RES). Avoiding RESclearance can allow the carriers to remain in the circulation longer,meaning that less of the associated drug need be administered to achievedesired serum levels. Enhanced circulation times can also allowtargeting of liposomes to non-RES containing tissues. The hydrophobicorganic moiety attached to the taxane can also be a headgroup-modifiedlipid.

The amount of the headgroup-modified lipid incorporated into the carrierdepends upon a number of factors well known to the ordinarily skilledartisan, or within his purview to determine without undueexperimentation. These include, but are not limited to: the type oflipid and the type of headgroup modification; the type and size of thecarrier; and the intended therapeutic use of the formulation. Theconcentration of the headgroup-modified lipid in the carrier isgenerally sufficient to prolong the circulatory half-life of the carrierin an animal, but is not so great as induce unwanted side effects in theanimal, and is typically at least about five mole percent of the lipidpresent in the carrier, The preferred headgroup-modified lipid isdipalmitoyl phosphatidylethanolamine-glutaric acid (DPPE-GA), which istypically used at a concentration of about 10 mole percent of the lipidpresent.

“Bioactive agent” as used herein denotes any compound or composition ofmatter which can be administered to animals and which can havebiological or diagnostic actiovity therein. Bioactive agents include,but are not limited to: antiviral agents such as acyclovir, zidovudineand the intereferons; antibacterial agents such as aminoglycosides,cephalosporins and tetracyclines; antifungal agents such as polyeneantibiotics, imidazoles and triazoles; antimetabolic agents such asfolic acid, purine and pyrimidine analogs; antineoplastic agents such asthe anthracycline antibiotics and plant alkaloids; such as cholesterol;carbohydrates, e.g., sugars and starches, amino acids, peptides,proteins such as cell receptor proteins, immunoglobulins, enzymes,hormones, neurotransmitters and glycoproteins; dyes; radiolabels such asradioisotopes and radioisotope-labelled taxanes; radiopaque taxanes;fluorescent taxanes; mydriatic taxanes; bronchodilators; localanesthetics; and the like. The additional bioactive agent used herein ispreferably an antimicrobial or antineoplastic agent. The additionalbioactive agent can be a therapeutic lipid, such as a ceramide. Theadditional agent can also be a second taxane derivative.

Further provided herein is a method of administering a taxane derivativeto an animal, e.g., a human. The method comprises administering to theanimal a composition comprising the derivative and a pharmaceuticallyacceptable medium. The medium preferably comprises a lipid carrier, morepreferably a liposome, associated with the taxane derivative. The taxanederivative used in the method of this invention comprises a hydrophobicorganic moiety attached to a taxane, and has the formula:

A¹ is H or a group having the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)—; Q isC₆H₅—, (CH₃)₃C—O— or (CH₃)CH═C(CH₃)—; A² is H or CH₃C(O)—; A³ is H orOH; R is H, or a group having the formula Y¹, Z¹X¹, or Z¹D¹; and R¹ isH, or a group having the formula Y², Z²X², or Z²D². When R¹ is H, A¹ isa group having the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)— and R is not H;when A¹ is H or when A¹ is a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)—R¹ is not H; and, at least one of R and R¹ isnot H. When R¹ is H, R is preferably a group having the formula Y¹. WhenA¹ is H, or when R is H, R¹ is preferably a group having the formula Y².

Each of Y¹ and Y² is independently a group having the formulaC(O)(CH₂)_(a)(CH═CH)_(b)(CH₂)_(c)(CH═CH)_(d)(CH₂)_(e)(CH═CH)_(f)(CH₂)_(g)(CH═CH)_(h) (CH₂)_(i)CH₃. The sum of a+2b+c+2d+e+2f+g+2h+i isequal to an integer 22; a is zero or an integer from 1 to 22; each of b,d, f and h is independently zero or 1; c is zero or an integer from 1 to20; e is zero or an integer from 1 to 17; g is zero or an integer from 1to 14; i is zero or an integer from 1 to 11; and a to i can be the sameor different at each occurrence. Each of Y¹ and Y² is preferably, andindependently, a group having the formula —C(O)(CH₂)_(a)CH₃. When R is agroup having the formula Y¹, and when R¹ is a group having the formulaY², each is independently preferably —C(O)(CH₂)₁₀CH₃ or C(O)(CH₂)₁₆CH₃.

Each of X¹ and X² is independently a group having the formula:

G¹ is —OP(O)₂OCH₂CH₂N(CH₃)₃, —OP(O)₂OCH₂CH₂NH₂, —OP(O)₂OCH₂CH(OH)CH₂OH,or —OP(O)₂OCH₂CH(NH₂)CO₂H.

Each of Z¹ and Z² is independently a linker of the formula:—C(O)(CH₂)_(j)(CH═CH)_(k)(CH₂)_(l)CH═CH)_(m)(CH₂)_(n)(CH═CH)OCH₂)_(p)(CH═CH)_(q)(CH₂)C(O)—. The sum of j+2k+l+2m+n+2o+p+2q+r isequal to an integer from 2 to 22; each of k, m, o and q is independentlyzero or 1; j is zero or an integer from 2 to 22; l is zero or an integerfrom 1 to 20; n is zero or an integer from 1 to 17; p is zero or aninteger from 1 to 14; r is zero or an integer from 1 to 11 and each of jto r can be the same or different at each occurrence. Preferably, eachof Z¹ and z² independently has the formula: —C(O)(CH₂)_(j)C(O)—; morepreferably, each of Z¹and Z² is —C(O)(CH₂)₈C(O)—.

Each of D¹ and D² is independently a group having the formula:

Preferably, each of Y¹ and Y² is then independently a group having theformula —C(O)(CH₂)_(a)CH₃, for example, —C(O)(CH₂)₁₄CH₃.

Animals afflicted with cancers can be treated according to the method ofthis invention, by administration of an anticancer effective amount of ataxane derivative provided herein. Paclitaxel derivatives are preferredfor use herein. Generally, those cancers treatable by the method of thisinvention are those which may be treated with the corresponding free,i.e., unattached taxane, and include, without limitation: carcinomas,myelomas, neuroblastomas, or sarcomas, of the brain, breast, lung,colon, prostate or ovaries, as well as leukemias or lymphomas.

Anticancer activity of taxane derivatives can be examined in vitro, forexample, by incubating a cancer cell culture with the derivative, andthen evaluating cell growth inhibition in the culture. Suitable cellsfor such testing include murine P388 leukemia, B16 melanoma and Lewislung cancer cells, as well as human mammary MCF7, ovarian OVCAR-3 andA549 lung cancer cells. GI₅₀ values, that is, the concentration of ataxane derivative required to induce 50% cell growth inhibition in aculture, for a derivative can be determined and compared. The lower ataxane derivative's GI₅₀, the lower is the amount of the derivativerequired to inhibit cancer cell growth. Accordingly, compounds withlower GI₅₀'s can have better therapeutic indices.

Alternatively, a taxane derivative can be tested in vivo for antitumoractivity, for example, by first establishing tumors in suitable testanimals, e.g., nude mice. Cells suitable for establishing tumors includethose described above for in vitro testing, as well as other cellsgenerally accepted in the art for establishing tumors. Subsequently, thetaxane derivative is administered to the animal; ED₅₀ values, that is,the amount of the derivative required to achieve 50% inhibition of tumorgrowth in the animal are then determined, as are survival rates.Ordinarily skilled artisans, given the teachings of this invention, arewell able to select particular taxane derivatives for applicationagainst certain cancers, on the basis of such factors as GI₅₀, ED₅₀ andsurvival values.

For the purposes of this invention, an “anticancer effective amount” ofa taxane derivative is any amount of the derivative effective toameliorate, lessen, inhibit or prevent the establishment, growth,metastasis, invasion or spread of a cancer. Anticancer effective amountsof taxane derivatives of this invention can be the same amount astherapeutic doses of the corresponding free taxane. However, theattachment of a hydrophobic organic moiety to the taxane so as to obtaina taxane derivative, and the association of this derivative with alipid, e.g., a liposome, in a carrier, can enhance the drug'stherapeutic index. Accordingly, this can mean that less of the taxanederivative, in comparison to the free taxane, need be used to achievethe desired therapeutic effect, and accordingly, that anticancereffective amounts of the derivative can be less than anticancereffective amounts of the free taxane.

Anticancer effective amounts of taxane derivatives can be chosen inaccordance with a number of factors, e.g., the age, size and generalcondition of the subject, the cancer being treated and the intendedroute of administration of the derivative, and determined by a varietyof means, for example, dose ranging trials, well known to, and readilypracticed by, ordinarily skilled artisans given the teachings of thisinvention. Generally, the anticancer effective amount of the taxanederivative of this invention is at least about 0.1 mg of the derivativeper kg of body weight of the animal to which the composition isadministered. Preferably, the anticancer effective amount of the taxanederivative is from about 0.1 mg per kg to about 1000 mg per kg.

Furthermore, the method provided herein can comprise administering anadditional bioactive agent, typically an antineoplastic agent, to theanimal. This additional bioactive agent can be administered to an animalprior to, concurrently with or subsequently to administration of thetaxane derivative of this invention. The additional agent can beentrapped in a liposome, for example, the same liposome with which thetaxane derivative of this invention can be associated.

This invention will be better understood from the following Examples.However, those of ordinary skill in the art will readily understand thatthese examples are merely illustrative of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1 Synthesis of Taxanes

2′ caproyl-paclitaxel

Paclitaxel (100 mg, 0.117 mmoles), dimethylaminopyridine (DMAP), (18 mg,0.133 mmoles), and 10 ml of chloroform were combined in an oven-dried,50-ml round-bottom flask with 18 mg (0.147 mmoles) of caproyl chloride,and incubated at room temperature. Reaction progress was monitored bysilica-based thin layer chromatography (TLC), using a 3%methanol-in-chloroform solvent system. By 4 hours, the spotcorresponding to paclitaxel (R_(f)=0.3) was no longer evident; a spotnot present in the analysis of the initial reaction mixture (R_(f)=0.5)was present.

Water (25 ml) was added to the reaction, and then extracted intochloroform to remove most of the DMAP. After drying with magnesiumsulfate, material from the chloroform phase was dissolved in 1% methanolin chloroform. The dissolved material was then added to a plug of silicaGel 60 (Fluka Fine Chemicals) 4 cm high×4 cm diameter, and 300 ml of a1% methanol-in-chloroform mixture was run through the plug.

The resulting compound was identified as 2′caproyl-paclitaxel(paclitaxel in which CH₃(CH₂)₄C(O)— was attached tothe 2′—OH group of paclitaxel) by NMR spectroscopy (see below).

7 Lauroyl-Paclitaxel

Paclitaxel (50 mg, 0.059 mmoles), DMAP (54 mg, 0.44 mmoles), lauroylchloride (77 mg, 0.35 mmoles), and 5 ml of chloroform were combined in a50 ml round-bottom flask, and incubated at room temperature. Reactionprogress was monitored as described above. At 4 hours of incubation, thespot corresponding to paclitaxel was no longer present, the spotcorresponding to 2′ lauroyl-paclitaxel (R_(f)=0.5) was the largest, andanother spot (R_(f)=0.7) began to appear. At 24 hours, the spotcorresponding to 2′ lauroyl-paclitaxel had disappeared, and the spot(R_(f)=0.7) corresponding to paclitaxel acetylated at both the 2′ and 7positions had increased in size.

The reaction was then extracted, and run through a silica plug, asdescribed above. Flash chromatography, using a solvent system comprising1.5% methanol-in-chloroform was used to separate the material at spotR_(f)=0.7 from the material running with the solvent front. The compoundwas identified as 2′,7 di-caprolypaclitaxel by NMR spectroscopy.

Fifty-eight mg (0.048 mmoles) of the diacetylated material was combinedwith 4.2 mg of sodium bicarbonate dissolved in 75 ul of water, in 30 mlof chloroform/methanol (1:1). Reaction progress was assayed frequently,so as to minimize hydrolysis of other ester linkages on the moleculewhile hydrolyzing the 12 carbon residue present at the 2′ position. At 8hours, one major peak (R_(f)=0.5), and two minor peaks, (R_(f)=0.55,0.45), were observed; however, most of the material in the reactionmixture appeared to be starting material. Further incubation did notsubstantially increase the major spot (R_(f)=0.5). Accordingly, 25 ml ofwater was added to the reaction mixture, and then extracted intochloroform. Preparative TLC chromatography (Whatman 20×20 c, fluorescentat @ 254 nm, 1000 micron plate) was used to purify the compounds. Thecompound was identified as 7 caproyl-paclitaxel by NMR spectroscopy.

NMR Spectroscopy

¹H and proton-decoupled ¹³C spectra of paclitaxel reacted with 6, 12 and18 carbon fatty acids were taken. Shifts in the resonance identified asbeing protons alpha to the hydroxyl groups are indicative of the ofacylation of the corresponding hydroxyl group (see, for example,Kingston, Pharm. Ther. 52 (1991), pp. 1-34). Reactions with the 2′ OHgroup proton are characterized by disappearance of resonance at 3.6 ppmand a shift of the proton alpha to the hydroxyl group from 4.8 ppm to5.6 ppm. Similar changes were reported by Kingston upon acetylation ofthe 2′ OH group.

Paclitaxel derivatized with fatty acids at the 2′ and 7 positions showthe same changes as occur with 2′ derivatization, as well as loss ofresonance at 2.5 ppm (7 OH group proton) and a resonance shift from 4.5ppm to 5.65 ppm. Reactions with the 7 hydroxy proton are characterizeddisappearance of resonance at 2.5 ppm, and a shift of the proton alphato the OH group from 4.5 to 5.65 ppm. Similar to changes were reportedby Kingston with acetylation of the 7 OH group¹. Carbon spectra ofpaclitaxel attached to a fatty acid have an additional resonance, infrequencies associated with the carbonyl functional group (165 to 200ppm), as well as resonances in frequencies associated with aliphaticcarbons (10 to 60 ppm).

Example 2

Association Assays

Liposomes were prepared, using the lipid concentrations indicated in thetable below (“DSPC”=distearoyl phosphatidylcholine; “EPC”=eggphosphatidylcholine; “GA”=dipalmitoyl phosphatidylethanolamine/GlutaricAcid; “Chol”=cholesterol; “drug”=taxane derivative and standardprocedures to make multilamellar liposomes; these were then extruded tentimes through a filter having 100-nm pores so as to obtain unilamellarliposomes (see Cullis et al., U.S. Pat No. 5,008,050).

Paclitaxel and hydrophobic derivatives of paclitaxel were formulated inliposomes composed of POPC. 54.6 nanomoles of the liposomal formulationof each drug was passed through a 55 cm×3 cm column filled with BioGelA-15m. 200-400 mesh Bio-Rad agarose beads, at a flow rate of 6 cm/min.

Results are presented in FIG. 1. For paclitaxel approximately 20% of thedrug remains associated with liposomes after passage through the column.However, for 7 caprolylpaclitaxel approximately 90% of the drug remainsassociated with liposomes.

Table 1, the “Summary of the Formulation Study Table,” presented belowis a summary of the results from several studies of the effect of theliposomal composition on 7 caproyl-paclitaxel and 7 stearoyl-paclitaxel.In particular, effects of saturation, cholesterol and PE-GA inclusionwere examined. Column 1 describes the liposomal formulation examined,column 2 the amount of drug associated with liposomes after passagethrough gel filtration columns (as described above), column 3 is aqualitative index of liposome aggregation (as observed by microscopy),and column 4 the mole percent of taxane associated with lipid afterliposome formation and extrusion through 100 nm filters. Unlessotherwise stated, initial paclitaxel concentration was 5 mole percent.

TABLE 1 SUMMARY OF FORMULATION STUDY % RETAINED % drug post FORMULATIONAvg. Std. Dev. AGG. extrusion 7 Stearoyl-paclitaxel DSPC; 10% GA; 5%Drug 49 1 — 4 EPC; 10% GA; 5% Drug 78 14 — 3 EPC; 25% CHOL; 5% Drug 12925 *** 4 7 caproyl-paclitaxel EPC; 20% GA; 15% Drug 56 10 — 12 EPC; 10%GA; 15%; Drug 44 7 — 14 EPC; 10% GA; 5% Drug 37 11 — 8 EPC; 25% CHOL; 5%Drug 31 1 ** 6 EPC; 25% CHOL; 10% GA; 43 11 * 6 5% Drug DSPC; 10% GA; 5%Drug 61 20 — 3 DSPC; 25% CHOL; 5% Drug 45 **** 1 DSPC; 25% CHOL, 10% GA;30 ** 1 5% Drug

Example 3

Characterization of Taxane-Containina POPC Lirosomes

Palmitoyloleoyl phosphatidylcholine (POPC) liposomes containing eitherpaclitaxel itself or a taxane-hydrophobic organic moiety conjugate wereprepared at a mole ratio of 95:5, lipid:drug according to proceduresdescribed hereinabove. The lipid, conjugate and paclitaxelconcentrations were determined after liposome preparation, as well asbefore and after passing through a size exclusion column (200-400 meshBio-Rad agarose beads). The decrease, if any, in the association ofpaclitaxel or the conjugate with the POPC was calculated and used as ameasure of association with liposomes. Results are presented in Table 2,below.

TABLE 2 LIPOSOME ASSOCIATION STUDIES Compound % Taxane Associated withLiposomes 2′-caproyl paclitaxel 40.8 2′-lauroyl paclitaxel 50.92′-stearoyl paclitaxel 65.7 7-caproyl paclitaxel 63.2 7-lauroylpaclitaxel 72.6 7-stearoyl paclitaxel 89.5 Paclitaxel 21.7

Example 4

In Vivo Studies

Liposomes were made, with dipalmitoyl phosphatidylcholine (DSPC),distearoyl phosphatidylethanolamine-glutaric acid (DPPE-GA) and7-caproyl paclitaxel (7-C6), according to procedures describedhereinabove at a molar ratio of DSPC:DPPE-GA:7-C6 of 8:1:0.6.

P-388 Studies

Groups of mice were injected with 1×10⁵ P388 cells and then, 24 hourslater, either Taxol® (cremophor-based paclitaxel suspension) or ataxane-containing liposome. “Plain,” that is, non-taxane-containing,liposomes were administered at 512 mg/kg of body weight, which wasequivalent to a 50 mg/kg dose of the taxane-containing liposomes.Liposome, or Taxol®, administration was repeated at days 2, 3, 4 and 5post cell administration. Days of death were then observed, and meansurvival times calculated. These data are presented in Table 3, below.

TABLE 3 SURVIVAL OF P388 TUMOR-BEARING MICE Mean Survival Time, ILS¹ asof Treatment Dose (mg/kg) Days (# mice) Control PBS² 0.2 ml 11.6 ± 0.2(5) — Plain Liposomes 512 mg/kg 11.2 ± 0.2 (5) (−)3.5 7-C6 Liposomes 5017.2 ± 0.7 (5) 48.2 7-C6 Liposomes 25  13.2 ± 0.2 (10) 13.8 Taxol ® 12.5 17.6 ± 0.8 (10) 51.7 ¹: “ILS” = increased life span = ((mean survivaltime (MST)treatment group/MST control) × 100) − 100; ²: phosphatebuffered saline.

B-16 Studies

Groups of mice were injected (intravenously) with 1×10⁵ B-16 cells, andthen, one day later, either PBS, plain liposomes, Taxol®, or 7-C6liposomes. Treatment was repeated on days 3, 5, 7 and 9 post-celladministration. On day 21, the animals were sacrificed, and their lungswere removed and fixed in formalin. The number of melanotic lung noduleswas counted “blind” using a magnifier; data is presented in Table 4,below.

TABLE 4 EFFECT ON DEVELOPMENT OF B16 MELANOMA LUNG TUMORS Mean Number of% Reduction in Tumor Nodules Nodule # vs. Treatment Dose (mg/kg) (#mice) PBS-Control PBS² 0.2 ml 27.1 ± 2.6 (8) — Plain Liposomes 512 mg/kg28.6 ± 1.4 (8) (−)5.5 7-C6 Liposomes 50 11.4 ± 1.2 (8) 57.9 7-C6Liposomes 25 13.3 ± 2.3 (8) 50.9 Taxol ® 12.5 17.5 ± 2.7 (8) 35.4

Example 5

Growth Inhibition Studies

Cells (A549, MCF7 or Lewis Lung) were incubated with variousconcentrations of paclitaxel; cell growth inhibition was determined bystandard means. Results of these experiments are presented in Tables 5and 6 (below), with the data there indicating the concentration (GI₅₀),micromolar, of either free paclitaxel, or a free paclitaxel derivative(Table 5) or liposomal paclitaxel or derivative (Table 6), that is, theconcentration found to inhibit about fifty percent of the growth of theindicated cell lines.

TABLE 5 GROWTH INHIBITION BY THE PACLITAXEL AND IT'S DERIVATIVES CellLine LEWIS Compound A549 MCF7 LUNG Paclitaxel 0.004 +/−  0.004 +/− 0.031 +/−   0.0001  0.0001 0.012 2′-C6 0.41 +/− 0.50 +/− 1.22 +/− 0.1340.151 0.592 2′-C12 0.45 +/− 0.84 +/− 1.26 +/− 0.08  0.17  0.87  2′-C187.56 +/− >6.0 >10 1.513 2′,7-diC6 >10 >10 >10 2′,7-diC12 >10 >10 >102′,7-diC18 —* — — 7-C6 0.032 +/−  0.027 +/−  0.091 +/−  0.002 0.0190.019 7-C12 4.71 +/− >10 7.89 +/− 0.25  0.37  7-C18 >10 >10 >10 *Notsoluble in DMSO or EtOH.

TABLE 5 GROWTH INHIBITION BY THE PACLITAXEL AND IT'S DERIVATIVES CellLine LEWIS Compound A549 MCF7 LUNG Paclitaxel 0.004 +/−  0.004 +/− 0.031 +/−   0.0001  0.0001 0.012 2′-C6 0.41 +/− 0.50 +/− 1.22 +/− 0.1340.151 0.592 2′-C12 0.45 +/− 0.84 +/− 1.26 +/− 0.08  0.17  0.87  2′-C187.56 +/− >6.0 >10 1.513 2′,7-diC6 >10 >10 >10 2′,7-diC12 >10 >10 >102′,7-diC18 —* — — 7-C6 0.032 +/−  0.027 +/−  0.091 +/−  0.002 0.0190.019 7-C12 4.71 +/− >10 7.89 +/− 0.25  0.37  7-C18 >10 >10 >10 *Notsoluble in DMSO or EtOH.

What is claimed is:
 1. A taxane derivative having the formula:

wherein: A¹ is H or a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)—; Q is C₆H₅—, (CH₃)₃C—O— or (CH₃)CH═C(CH₃)—;A² is H or CH₃C(O)—; A³ is H or OH; R is H, a saturated or unsaturatedfatty acid moiety, or a group having the formula Y¹, Z¹X¹, or Z¹D¹; R¹is H, a saturated or unsaturated fatty acid moiety, or a group havingthe formula Y², Z²X², or Z²D²; wherein each of X¹ and X² isindependently a group having the formula:

wherein G¹ is —OP(O)₂OCH₂CH₂N(CH₃)₃,—OP(O)₂OCH₂CH₂NH₂,—OP(O)₂OCH₂CH(OH)CH₂OH, or —OP(O)₂OCH₂CH(NH₂)CO₂H;wherein each of D¹ and D² is independently a group having the formula:

wherein: each of Y¹and Y² is independently an acyl chain having 7 to 24carbon atoms; a group having the formulaC(O)(CH₂)_(a)(CH═CH)_(b)(CH₂)_(c)(CH═CH)_(d)(CH₂)_(e)(CH═CH)_(f)(CH₂)_(g)(CH═CH) _(h)(CH₂)_(i)CH₃, the sum ofa+2b+c+2d+e+2f+g+2h+i is equal to an integer of from 7 to 22, a is zeroor an integer from 1 to 22, each of b, d, f and h is independently zeroor 1, c is zero or an integer from 1 to 20, e is zero or an integer from1 to 17, g is zero or an integer from 1 to 14, i is zero or an integerfrom 1 to 11, a to i can be the same or different at each occurrence;wherein: each of Z¹ and Z² is independently a linker of the formula:—C(O)(CH₂)_(j)(CH═CH)_(k)(CH₂)_(l)(CH═CH)_(m)(CH₂)_(n)(CH═CH)_(o)(CH₂)_(p)(CH═CH)_(q)(CH₂)_(r)C(O)—, the sum ofj+2k+l+2m+n+2o+p+2q+r is equal to an integer from 2to 22, each of k, m,o and q is independently zero or 1, j is zero or an integer from 2 to22, l is zero or an integer from 1 to 20, n is zero or an integer from 1to 17, p is zero or an integer from 1 to 14, r is zero or an integerfrom 1 to 11, each of j to r can be the same or different at eachoccurrence. wherein when R¹ is H, A¹ is a group having the formulaQ—C(O)NHCH(C₆H₅)CH(OR)C(O)— and R is not H; wherein when A¹ is H or whenA¹ is a group having the formula Q—C(O)NHCH(C₆H₅)CH(OR)C(O)— and R is H,R¹ is not H; and wherein at least one of R and R¹ is not H.
 2. Thetaxane derivative of claim 1, wherein at least one of R and R¹ is anunsaturated fatty acid moiety.
 3. The taxane derivative of claim 2,wherein at least one of R and R¹ is an aliphatic fatty acid moiety. 4.The taxane derivative of claim 3, wherein R is an unsaturated aliphaticfatty acid moiety, and R¹ is H.
 5. The taxane derivative of claim 4,wherein Q is C₆H₅—.
 6. The taxane derivative of claim 5, wherein A² isCH₃C(O)— and wherein A³ is H.
 7. The taxane derivative of claim 1,wherein at least one of R and R¹ is a group having the formulaC(O)(CH₂)_(a)(CH═CH)_(b)(CH₂)_(c)(CH═CH)_(d)[(CH₂)_(e)(CH═CH)_(f)]_(s)(CH₂)_(g)CH₃, the sum of a+2b+c+2d+(e+2f)s+g is equal toan integer of from 7 to 22, a is zero or an integer from 1 to 22, eachof b, d, and f is independently zero or 1, c is zero or an integer from1 to 20, e is zero or an integer from 1 to 17, s is zero or an integerfrom 1 to 7, g is zero or an integer from 1 to 14, and a to g can be thesame or different at each occurrence.
 8. The taxane derivative of claim7, wherein R¹ is H.
 9. The taxane derivative of claim 8, wherein Q isC₆H₅.
 10. The taxane derivative of claim 9, wherein A² is CH₃C(O)— andwherein A³ is H.